This invention relates to minimised ribozymes, herein referred to as xe2x80x9cminiribozymesxe2x80x9d, and in particular it relates to a class of miniribozymes which has been selected on the basis of the very high cleavage rates by the members of the class at low Mg2+ concentration. This invention also extends to compositions comprising these miniribozymes, to transfer vectors and host cells as well as methods of cleaving a target RNA in a subject and other methods of use of these miniribozymes.
Throughout this application, various references are cited in brackets. The full citations may be found in the numerical list of references immediately following the Example(s). These publications are hereby incorporated by reference into the present application.
There is currently much interest in the catalytic potential of RNA both as a candidate molecule for precellular evolution and as gene targeted therapeutics. To date there are in excess of 30 distinct RNA motifs known to perform catalysis in the absence of protein. These include ribozymes derived from natural RNAs and also those produced by in vitro selection (1). The reactive capacity of any polynucleotide is a function of the chemistry of the individual nucleotides and the secondary and tertiary structure of the polymer, both of which are affected by coordination with metal ions (2,3). Structure within an oligonucleotide can be viewed as a direct function of the sequence identity. The catalytic versatility of a nucleic acid is therefore amenable to combinatorial studies, such as in vitro selection, involving the artificial manipulation of a discrete sequence space in response to selective pressure.
The 2xe2x80x2 hydroxyl of ribonucleic acids is a crucial functional entity permitting nucleophilic attack by the deprotonated form on an adjacent bridging phosphate ester leading to hydrolysis (giving 5xe2x80x2 OH and 2xe2x80x2-3xe2x80x2 cyclic phosphate ends) via a pentacoordinate transition state (4-7). Enzymes of either nucleic acid or protein composition that catalyse reactions of phosphates are invariably metal ion dependent (8). Metals have been recruited by biology for this purpose presumably because phosphates are a favourable metal ion ligand, and because coordination with metals withdraws electron density from the phosphorus centre, making it more susceptible to nucleophilic attack. Metal ions may also assist the deprotonation of the attacking nucleophile and assist the stabilisation of the developing negative charge on the leaving group (8-10).
The hammerhead ribozyme was first identified as a self (cis) cleaving sequence found in a number of small, circular, RNA pathogens (virusoids and viroids) found in plants, and a satellite RNA found in newt (11). Its consensus structure consists of three helical regions which form at their junction, a conserved bed of 15 nucleotides. The bulk of the conserved nucleotides can be located on a single oligoribonucleotide constituting an enzymatic entity capable of cleaving multiple substrates (12,13). Ribozymes designed accordingly can be directed in trans against any RNA substrate containing an endogenous 5xe2x80x2 UH (where H=C, U, or A) (14-16). There is significant interest in these enzymes because they offer a means of specifically inactivating deleterious RNA, eg. viral or oncogenic mRNAs, and thereby ameliorating disease.
Hammerhead cleavage of an RNA phosphodiester bond exhibits divalent metal ion dependence (17) typical of this form of catalysis in nature. Generally the metal ion is proposed to act both structurally to augment and direct specific helical interactions, and as a catalytic co-factor functional in the chemical step of the reaction (2,9,18). Mg2+ is known to perform both these roles effectively. Bassi et al (19-21) report that there is a two stage folding process leading to formation of the ground state, each with a specific Mg2+ requirement. Once the ground state is obtained, Mg 2+ is thought to play a role in activating the 2xe2x80x2 OH nucleophile of C17, either by direct coordination, or by providing an appropriately positioned basic group (Mgxe2x80x94OH), to effect deprotonation, promoting nucleophilic attack on the adjacent phosphorus (22-24).
The role of helix II in the hammerhead ribozyme has been investigated in several deletion studies (25-28). The presence of Watson-Crick base pairing in this region is thought to stabilise the active conformation of the conserved nucleotides stacked above it (28). The role of helix II is therefore seemingly to limit the number of steric possibilities closer to the cleavage site. This effect is no doubt variable and will depend on the composition of nucleotides between positions A9 and G12. Crystal structures for the hammerhead ribozyme show proximity between helix I and helix II (29,30). These structures suggest a plausible interaction (perhaps Mg2+ mediated) between helix I and helix II. Whilst this interaction is remote from the cleavage site, it affects the global architecture of the molecule, and thus the cleavage rate. It has been speculated that the interaction between helix I and II may in fact stabilise an inactive conformation (31). The truncation of helix I appears to amend this interaction and allows higher rates of catalysis to be observed (31). Minimisation strategies involving helix II therefore might offer an alternative means of circumventing or closing down this particular equilibrium pathway, and thereby improve the catalytic outcome.
Miniribozymes are derivatives of the hammerhead ribozyme where helix II has been replaced by a linker with a single Watson-Crick base pair (32). This minimisation strategy has created a novel structural format. Whilst the full length ribozyme has been subject to selection over evolutionary time, size constrained, trans cleaving ribozymes have not been exposed to selection in nature. Minimised ribozymes have been shown to cleave long RNAs more efficiently than full length hammerheads (27), and could therefore provide improved trans cleaving activity in a cellular environment.
The work leading to the present invention has included in vitro optimisation of the novel miniribozyme structure. In particular, in vitro selection was used to search an 18 nt RNA sequence space corresponding in size to a miniribozyme (FIG. 1). The aims were primarily to identify all motifs within this size constrained domain, capable of supporting Mg2+ dependent phosphodiester cleavage of a 29 mer RNA substrate containing a 13 nt segment of human IL-2 mRNA. Subsequently, the aim was to direct the active component of this population towards optimum catalytic efficiency at low concentrations of Mg2+ (0.5-2 mM) such as occur intracellularly (33). This work has shown that the active population consisted almost entirely of molecules containing conserved nucleotides conforming to recognised hammerhead motifs. This set of molecules exhibited highly variable catalytic activity. It was an expectation that hammerhead-like molecules would form a subset of the active sequence space. An important goal was therefore to optimise the nucleotide composition between positions 9 and 12 amongst hammerhead-like molecules, within a context of size constraint, and therefore to evolve a linker between A9 and G12 which most efficiently favours equilibration of the active conformation.
This invention is directed to a selected class of miniribozymes, capable of hybridising with a target RNA to be cleaved, and exhibiting very high cleavage rates at low Mg2+ concentration. These miniribozymes may be used both in vitro and in vivo, and their uses extend to both the diagnostic and therapeutic fields, that is, these miniribozymes may be used as diagnostic or therapeutic agents.
In one aspect, the present invention provides a compound of the formula IA (SEQ ID NO:1) or 1B (SEQ ID NO:2): 
wherein each X represents a nucleotide which may be the same or different and may be substituted or modified in its sugar, base or phosphate; and wherein G10.1 and C11.1 each represent a nucleotide which may be substituted or modified in its sugar (which may be ribose or deoxyribose), base or phosphate;
wherein each of C, G, A and U represents a ribonucleotide which may be substituted or modified in its sugar, base or phosphate;
wherein each of (X)n and (X)nxe2x80x2 represents an oligonucleotide having a pre-determined sequence which is capable of hybridizing with an RNA target sequence to be cleaved, such RNA target sequence not being present within the compound, and each of n and nxe2x80x2 represents an integer which defines the number of nucleotides in the oligonucleotide;
wherein Xxe2x80x2 represents a ribonucleotide selected from C, G, A and U which may be substituted or modified in its sugar, base or phosphate;
wherein a defines the number of nucleotides in (X)a and may be 0 or 1 and if 0, the A located 5xe2x80x2 of (X)a is bonded to the G located 3xe2x80x2 of (X)a;
wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof;
wherein each N represents a nucleotide selected from C, G, A and U/T which may be substituted or modified in its sugar (which may be ribose or deoxyribose), base or phosphate and wherein each H represents a nucleotide selected from C, A and U/T, which may be substituted or modified in its sugar (which may be ribose or deoxyribose), base or phosphate; with the proviso that the sequence 5xe2x80x2-NNHH-3xe2x80x2 is not UUUU or TTTT, CUCC, AAUU or GGCA.
In formulae IA and 1B, C represents a nucleotide in which the base is cytosine, G represents a nucleotide in which the base is guanine, A represents a nucleotide in which the base is adenine, U represents a nucleotide in which the base is uracil, and T represents a nucleotide in which the base is thymine.
In one embodiment of the present invention, the oligonucleotide 3xe2x80x2-(X)n- in the compound of formula I is 3xe2x80x2-(X)n-1-A-.
The compounds of formula IA as set out above, include the linker sequence 5xe2x80x2-NNHH-3xe2x80x2 which is preferably selected from the following classes of linker sequences:
Class I: YRHH, wherein Y is C or U, R is G or A, and H is C, A or U.
Class II: DYHH, wherein D is G, A or U, Y is C or U, and H is C, A or U.
Class III: GHHA, wherein H is C, A or U.
Preferred linker sequences in Class I are the sequences CGUU, UGUU and UAAC. Preferred linker sequences in Class II are sequences of the class WYHH (wherein W is A or U, Y is C or U, and H is C, A or U), particularly the sequences ACCC, AUUU, UCCC, AUUC, AUUA, ACAC, AUAA and AUAC. Particularly preferred linker sequences in Class II are the pyrimidine rich subclass of sequences UUHH, wherein H is C, A or U, in particular the sequences UUAC, UUCC, UUUC, UUUA, UUAA and UUAU. Preferred linker sequences in Class III are the sequences GUAM and GAUA.
The compounds of formula 1B as set out above include the linker sequence 5xe2x80x2-HNHHH-3xe2x80x2 which is preferably selected from the sequences UCCCA, UCCCC, UCCUA, AAUUU, UUAAA, UUUUA, UGUCC, UGUUA and CACCC. Particularly preferred sequences are UCCCC, UGUCC and CACCC.
While the preferred linker sequences described above are described as RNA sequences, it will be understood that these preferred linker sequences also include the corresponding DNA sequences.
In general terms, the compounds of the present invention are synthetic, non-naturally occurring oligonucleotide compounds comprising a sequence of nucleotides which includes a catalytic region and hybridizing regions whose sequences are capable of hybridizing with a predetermined RNA target sequence to be cleaved.
The present invention is also directed to compositions comprising a compound of formula IA or 1B above in association with an acceptable carrier.
The invention is also directed to an oligonucleotide transfer vector containing a nucleotide sequence which on transcription gives rise to a compound of formula IA or 1B above. The transfer vector may be a bacterial plasmid, a bacteriophage DNA, a cosmid, an eukaryotic viral DNA, a plant DNA virus, a composite geminivirus, a binary plant expression vector (Ri or Ti), an infective phage particle or a portion thereof. The packaged oligonucleotide transfer vector may contain promoter sequences for RNA polymerase II, human tRNAval, plant tRNA, human tRNA, snRNA promoter or RNA polymerase III. The invention also includes a host cell transformed by the transfer vector. The host cell may be a prokaryotic host cell or an eukaryotic host cell, such as an E. coli host cell, a monkey COS host cell, a Chinese hamster ovary host cell, a mammalian host cell, a plant host cell, a plant protoplast host cell, a hematopoietic host cell, a stem cell, a hematopoietic progenitor cell, a lymphoid cell, a T-cell, a B-cell, pre-B cell, a CD4+T-cell or a peripheral blood mononuclear cell.
The invention also provides a method of cleaving a target mRNA in a subject which comprises administering to the subject an effective amount of a compound of formula IA or 1B above or a vector capable of expressing the compound. The administration may be topical in an amount between 1 ng and 10 mg. The administration may also be systemic and administered in an amount between 1 ng and 500 xcexcg/kg weight/day. The administration may also be aerosol administration.
The invention also provides a method of cleaving a target mRNA in a host cell which comprises administering to the host cell an effective amount of a compound of formula IA or 1B or a vector capable of expressing the compound.
The compound of formula IA or 1B may further comprise an antisense nucleic acid which is capable of hybridizing with an RNA target sequence. The compound may further comprise at least one additional non-naturally occurring oligonucleotide compound which comprises nucleotides whose sequence defines a conserved catalytic region and nucleotides whose sequence is capable of hybridizing with a predetermined target sequence. The additional non-naturally occurring oligonucleotide compound may be a hammerhead ribozyme, a miniribozyme, a hairpin ribozyme, a hepatitis delta ribozyme, an RNAase P ribozyme, a Group I intron, or a combination thereof. See for example: hammerhead ribozyme (Haseloff et al. U.S. Pat. No. 5,254,678, issued Oct. 18, 1993; Jennings U.S. Pat. No. 5,298,612, issued Mar. 29, 1994); Group I introns, (Cech et al. U.S. Pat. No. 4,740,463, issued Apr. 26, 1988; Altman et al. U.S. Pat. No. 5,168,053, issued Dec. 1, 1992 or PCT International Publication No WO 92/03566); hepatitis delta ribozymes (PCT International Application No. WO 90/05157) and hairpin ribozymes (European Patent Application No. EP 360,257).
Preferred cleavage sites in the target RNA have the sequence xe2x80x9cNUHxe2x80x9d (wherein N represents C, G, A or U and H represents C, A or U), preferably GUC, GUU, GUA, UUA and UUC. By way of example, suitable reaction conditions may comprise a temperature from about 4xc2x0 C. to about 60xc2x0 C., preferably from about 10xc2x0 C. to 45xc2x0 C., more preferably from about 20xc2x0 C. to 43xc2x0 C.; pH from about 6.0 to about 9.0 and concentration of divalent cation (such as Mg2+) from about 0.1 to about 100 mM, preferably from about 1 to about 100 mM (most preferably 1 to 20 mM). The nucleotides of the sequences (X)n and (X)nxe2x80x2 of the compounds above may be of any number and sequence sufficient to enable hybridization with the nucleotides in the target RNA, as described herein. Ribozymes containing a small number of nucleotides in each of the groups (X)n and (X)nxe2x80x2 of the compounds above (such as four nucleotides) would generally be incubated at lower temperatures, such as about 20xc2x0 C. to about 25xc2x0 C. to aid hybridizing with the nucleotide sequences in the substrate. The number of nucleotides n and nxe2x80x2 in (X)n and (X)nxe2x80x2 are not necessarily equal. Preferably, the sum of n+nxe2x80x2 is greater than 14.
The invention is also directed to covalently-linked multiple ribozymes, where each ribozyme is directed to a target sequence which may be the same or different. In addition these compounds may be covalently attached to an antisense molecule which may be 10 to 100 bases in length. Antisense sequences capable of hybridizing to an RNA in a mammal or plant are well known (see Shewmaker et al. U.S. Pat. No. 5,107,065, issued Apr. 21, 1992). As the ribozyme acts as an enzyme, showing turnover, the ratio of ribozyme to substrate may vary widely.
A target RNA containing a suitable cleavage site such as an NUH site as described above may be incubated with a compound described above. The nucleotide sequences (X)n and (X)nxe2x80x2 of the compounds above are selected to hybridize with their substrate. They may be selected so as to be complementary to nucleotide sequences flanking the cleavage site in the target RNA. On incubation of the ribozyme or ribozyme composition and its substrate, an enzyme/substrate complex is formed as a result of base pairing between corresponding nucleotides in the ribozyme and the substrate. Nucleotide sequences complementary to (X)nxe2x80x2 and (X)nxe2x80x2 of the compounds above flanking the cleavage site in the substrate may form a double stranded duplex with (X)n  and (X)nxe2x80x2 as a result of base pairing, which base pairing is well known in the art (see for example: Sambrook, 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press). The formation of a double stranded duplex between the nucleotides may be referred to as hybridization (Sambrook, 1989). The extent of hybridization or duplex formation between the ribozyme and its substrate can be readily assessed, for example, by labelling one or both components, such as with a radiolabel, and then subjecting the reaction mixture to polyacrylamide gel electrophoresis under non-denaturing conditions (Sambrook, 1989). If the target is cleaved specifically on incubation with the compound, the compound is active and falls within the scope of this invention. Accordingly, a ribozyme containing substituted or modified nucleotides in the conserved region may be simply tested for endonuclease activity in a routine manner.
As will be readily appreciated by workers in the field to which this invention relates, the cleavage of a target RNA may be readily assessed by various methods well known in the art (see for example: Sambrook, 1989). Cleavage may, for example, be assessed by running the reaction products (where the substrate is radioactively labelled) on acrylamide, agarose, or other gel systems under denaturing conditions, and then subjecting the gel to autoradiography or other analytical technique to detect cleavage fragments (Sambrook 1989).
The invention is also directed to an oligonucleotide transfer vector containing a nucleotide sequence or sequences which on transcription gives rise to a compound of formula IA or 1B above. The transfer vector may be a bacterial plasmid, a recombinant bacterial plasmid, a bacteriophage DNA, a cosmid, or an eukaryotic viral DNA. The transfer vector may also contain an appropriate transcription promoter sequence such as that for RNA polymerase II, RNA polymerase III, a viral promoter such as SV40 or HIV LTR, a plant promoter such as CaMV S35 or a promoter associated with animal health. The vector may also contain an appropriate termination sequence. Preferably, the plant or animal promoter is capable of expression in a regulated manner. Such promoter control regions would be regulated by endogenous signals to direct either tissue specific or temporal expression, or by externally administered compounds to elicit transcription of downstream sequences. It may also contain sequences to effect integration into the host genome or episomal replication in the host cell.
The invention also provides a host cell transformed by the transfer vector as mentioned above, which may be a prokaryotic host cell or an eukaryotic host cell e.g. a yeast cell or yeast protoplast, E. coli host cell, a monkey host cell (e.g. COS), a Chinese hamster ovary host cell, a mammalian host cell, a plant host cell, or a plant protoplast host cell.
In one embodiment, there is provided a packaged oligonucleotide transfer vector, as mentioned hereinabove, which is a plant virus, a composite mammalian virus, a geminivirus, a Ti or Ri plasmid, an infective phage particle or portion thereof.
The nucleotides X in the compounds of formula IA or 1B may be in the form of deoxyribonucleotides, ribonucleotides, deoxyribonucleotide/ribonucleotide hybrids, or derivatives thereof as herein described.
The flanking sequences (X)n and (X)nxe2x80x2 in the compounds of formula IA or 1B may be chosen to optimize stability of the ribozyme from degradation. For example, deoxyribonucleotides are resistant to the action of ribonucleases. Modified bases, sugars or phosphate linkages of nucleotides, such as phosphoramidate or phosphorothioate linkages in the sugar phosphate chain of (X)n and (X)nxe2x80x2 may also provide resistance to nuclease attack. Binding affinity may also be optimized in particular circumstances, by providing nucleotides solely in the form of ribonucleotides, deoxyribonucleotides, or combinations thereof. In some circumstances it may be necessary to optimize the composition of the sequences (X)n and (X)nxe2x80x2 to maximize target RNA cleavage. The cleavage activity of ribozymes having flanking nucleotide sequences which hybridize to target sequences and which are comprised wholly of deoxyribonucleotides may, in some circumstances, have reduced activity. In such circumstances optimization may involve providing a mixture of deoxyribonucleotides and ribonucleotides in the nucleotide sequences (X)n and (X)nxe2x80x2. For example, nucleotides in the ribozyme which are proximal to the cleavage site in a target RNA may be in the form of ribonucleotides.
The respective 3xe2x80x2 and 5xe2x80x2 termini of the sequences (X)n and (X)nxe2x80x2 or alternatively the 3xe2x80x2 and 5xe2x80x2 end termini of the ribozyme, may be modified to stabilize the ribozyme from degradation. For example, blocking groups may be added to prevent terminal nuclease attack, in particular 3xe2x80x2-5xe2x80x2 progressive exonuclease activity. By way of example, blocking groups may be selected from substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted alkanoyl groups. Substituents may be selected from C1-C5 alkyl; halogens such as F, Cl or Br; hydroxy; amino; C1-C5 alkoxy and the like. Alternatively, nucleotide analogues such as phosphorothioates, methylphosphonates or phosphoramidates, or nucleoside derivatives (such as alphaxe2x80x94anomer of the ribose moiety) which are resistant to nuclease attack may be employed as terminal blocking groups. The blocking group may be an inverted linkage such as a 3xe2x80x23xe2x80x2 thymidine linkage or a 5xe2x80x25xe2x80x2 pyrophosphate linkage as in the guanosine cap.
Alternatively, groups which alter the susceptibility of the ribozyme molecule to nucleases may be inserted into the 3xe2x80x2 and/or 5xe2x80x2 end of the ribozyme. For example, 9-amino-acridine attached to the ribozyme may act as a terminal blocking group to generate resistance to nuclease attack on the ribozyme molecules and/or as an intercalating agent to aid endonucleolytic activity. It will be readily appreciated that a variety of other chemical groups, e.g. spermine or spermidine, could be used in a related manner.
It is also possible to stabilize the ribozyme from degradation by embedding it in an RNA molecule. These molecules can be produced either in vitro or in vivo by DNA coding sequences being operably linked to transcriptional control sequences as appropriate. Examples of RNA molecules into which ribozymes could be inserted may include, but are not limited to, tRNA, mRNA, rRNA, snRNA or other RNA molecules. In addition, the ribozyme may be inserted into an engineered stable stem loop structure. The compound may also be coupled with other stabilizing structures such as a transcription terminator on the 3xe2x80x2 end such as the T7 terminator, p-independent terminator, cry element (Gelfand et al. U.S. Pat. No. 4,666,848, issued May 19, 1987) or the TrpE terminator. Furthermore, sequences such as the poly(A) addition signal AAUAAA may be added. In addition, strategies involving changing the length of the 3xe2x80x2 noncoding region may be used (see Gillies, U.S. Pat. No. 5,149,635, issued Sep. 22, 1992). Alternatively, a stabilizing sequence or protein binding domain (see PCT International application WO 94/10301) may be used. Further, it is possible to insert the compound into a DNA molecule as well.
The compounds of this invention may be covalently or non-covalently associated with affinity agents such as proteins, steroids, hormones, lipids, nucleic acid sequences, intercalating molecules (such as acridine derivatives, for example 9-amino acridine) or the like to modify binding affinity for a substrate nucleotide sequence or increase affinity for target cells, or localization in cellular compartments or the like. For example, the ribozymes of the present invention may be associated with RNA binding peptides or proteins which may assist in bringing the ribozyme into juxtaposition with a target nucleic acid such that hybridization and cleavage of the target sequence may take place. Nucleotide sequences may be added to the respective 3xe2x80x2 and 5xe2x80x2 termini of the sequences (X)n and (X)nxe2x80x2 or alternatively the 3xe2x80x2 and 5xe2x80x2 end termini of the ribozyme to increase affinity for substrates. Such additional nucleotide sequences may form triple helices with target sequences which may enable interaction with an intramolecularly folded substrate. Alternatively, modified bases (for example, non-natural or modified bases as described in Saenger, 1984, Principles of Nucleic Acid Structure, Springer-Verlag N.Y.) within the additional nucleotide sequences may be used that will associate with either single stranded or duplex DNA generating base pair, triplet, or quadruplet, interactions with nucleotides in the substrate. Suitable bases would include inosine, 5-methylcytosine, 5-bromouracil and other such bases as are well known in the art, as described, for example, in Saenger, 1984.
The compounds of this invention may be produced by oligonucleotide synthetic techniques which are known in the art. Generally, such synthetic procedures involve the sequential coupling of activated and protected nucleotide bases to give a protected nucleotide chain, whereafter protecting groups may be removed by suitable treatment. Preferably the compounds will be synthesized on an automated synthesizer such as those made by Applied Biosystems (a Division of Perkin Elmer), Pharmacia or Millipore. Alternatively, the ribozymes in accordance with this invention may be produced by transcription of nucleotide sequences encoding said ribozymes in host-cells or in cell free systems utilizing enzymes such as T3, SP6 or T7 RNA-polymerase.
In addition to being synthesized chemically, ribozymes with modified nucleotides may be synthesized enzymatically. The phosphodiester bonds of RNA can be replaced by phosphorothioate linkages by in vitro transcription using nucleoside xcex1-phosphorothiotriphosphates. T7 RNA polymerase specifically incorporates the Sp isomer of xcex1-phosphorothiotriphosphate with inversion of configuration to produce the Rp isomer of the phosphorothioate linkage. Similarly, T7 RNA polymerase is also able to incorporate 2xe2x80x2 O modified nucleotide triphosphates, including 2xe2x80x2 O-methyl, 2xe2x80x2 O-fluoro and 2xe2x80x2 amino modified nucleoside triphosphates.
Nucleotides represented in the compounds for formula I above comprise a sugar, base, and a monophosphate group or a phosphodiester linkage. Accordingly, nucleotide derivatives or modifications may be made at the level of the sugar, base, monophosphate groupings or phosphodiester linkages. It is preferred that the nucleotides in the compounds above be ribonucleotides or RNA/DNA hybrids, however, other substitutions or modifications in the nucleotide are possible providing that endonuclease activity is not lost.
In one aspect of this invention, the sugar of the nucleotide may be a ribose or a deoxyribose such that the nucleotide is either a ribonucleotide or a deoxyribonucleotide, respectively. Furthermore, the sugar moiety of the nucleotide may be modified according to well known methods in the art (see for example: Saenger, 1984) This invention embraces various modifications to the sugar moiety of nucleotides as long as such modifications do not abolish cleavage activity of the ribozyme. Examples of modified sugars include replacement of secondary hydroxyl groups with halogen, amino or azido groups; 2xe2x80x2-alkylation; conformational variants such as the 2xe2x80x2-hydroxyl being cis-oriented to the glycosyl C1-N link to provide arabino-nucleosides, and conformational isomers at carbon C1 to give alpha-nucleosides, and the like. In addition, the invention is directed to compounds with a substituted 2xe2x80x2-hydroxyl, such as 2xe2x80x2 O-allyl or 2xe2x80x2 O-methyl. Alternatively, the carbon backbone of the sugar may be substituted, such as in 2xe2x80x2 C-allyl.
Accordingly, the base of the nucleotide may be adenine, 2-amino adenine, cytosine, guanine, hypoxanthine, inosine, methyl cytosine, thymine, xanthine, uracil, or other modified bases.
Nucleotide bases, deoxynucleotide bases, and ribonucleotide bases are well known in the art and are described, for example in Saenger, (1984). Furthermore, nucleotide, ribonucleotide, and deoxyribonucleotide derivatives, substitutions and/or modifications are well known in the art (see for example: Saenger, 1984); and these may be incorporated in the ribozyme made with the proviso that endonuclease activity of the ribozyme is not lost. As mentioned previously, endoribonuclease activity may be readily and routinely assessed.
In addition, a large number of modified bases are found in nature, and a wide range of modified bases have been synthetically produced (see for example: Saenger, 1984). For example, amino groups and ring nitrogens may be alkylated, such as alkylation of ring nitrogen atoms or carbon atoms such as N1 and N7 of guanine and C5 of cytosine; substitution of keto by thioketo groups; saturation of carbon-carbon double bonds, and introduction of a C-glycosyl link in pseudouridine. Examples of thioketo derivatives are 6-mercaptopurine and 6-mercaptoguanine. Bases may be substituted with various groups, such as halogen, hydroxy, amine, alkyl, azido, nitro, phenyl and the like.
The phosphate moiety of nucleotides or the phosphodiester linkages of oligonucleotides are also subject to derivatization or modifications, which are well known in the art. For example, replacement of oxygen with nitrogen, sulphur or carbon gives phosphoramidates, phosphorothioates or phosphorodithioates, and phosphonates, respectively. Substitutions of oxygen with nitrogen, sulphur or carbon derivatives may be made in bridging or non bridging positions. It has been well established from work involving antisense oligonucleotides that phosphodiester and phosphorothioate derivatives may efficiently enter cells (particularly when of short length), possibly due to association with a cellular receptor. Methylphosphonates are readily taken up by cells probably by virtue of the electrical neutrality.
A further aspect of the invention provides alternative linkages such as an amide, a sulfonamide, a hydroxylamine, a formacetal, a 3xe2x80x2-thioformacetal, a sulfide, or an ethylene glycol function to replace the conventional phosphodiester linkage. These modifications may increase resistance towards cellular nucleases and/or improve pharmacokinetics.
Detailed information on synthesis of protected nucleotides and their incorporation into modified ribozymes is provided in International Patent Application No. PCT/AU96/00343 (WO 96/40806), the contents of which are incorporated by reference into the present application.
Possible nucleotide modifications include the following, by way of example:
Sugar modifications may be 2xe2x80x2 fluoro, 2xe2x80x2 amino, 2xe2x80x2 O-allyl, 2xe2x80x2 C-allyl, 2xe2x80x2 O-methyl, 2xe2x80x2 O-alkyl, 4xe2x80x2-thio-ribose, xcex1-anomer, arabinose, other sugars, or non-circular analogues.
Phosphate modifications may be phosphorothioate (non-bridging), phosphorodithioate (non-bridging), 3xe2x80x2 bridging phosphorothioate, 5xe2x80x2 bridging phosphorothioate, phosphoramidate, 3xe2x80x2 bridging phosphoramidate, 5xe2x80x2 bridging phosphoramidate, methyl phosphonate, other alky/phosphonates or phosphate triesters
The phosphodiester linkage may be replaced by an amide, carbamate, thiocarbamate, urea, amine, hydroxylamine, formacetal, thioformacetal, allyl ether, allyl, ether, or thioether linkage. Alternatively, the phosphodiester linkage and the ribose may be replaced by the amide backbone in a PNA (peptide nucleic acid) linkage.
Base modifications may be purine, 2,6-diaminopurine, 2-aminopurine, 06-methylguanosine, C-5-alkenylpyrimidine derivatives, C-5-propynylpyrmidine derivatives, inosine, 5-methylcytosine, pseudouridine, abasic (ribose or deoxyribose).
Some nucleotides may be replaced with the following chemical linkers: 1,3-propane-diol, alkane-diols, or various polymers of ethyleneglycol, tetraethylene glycol or hexaethyleneglycol.
Other modifications to the 3xe2x80x2 end may be selected from: 3xe2x80x2-3xe2x80x2 inverted linkage (inverted nucleotide or inverted abasic nucleotide), 3xe2x80x2-3xe2x80x2 linked abasic ribose, or end-capped (methoxyethylamine phosphoramidate).
International Patent Application No. PCT/AU96/00343 sets out in detail methods by which these modified nucleotides may be synthesised, as well as nucleotide modifications which have been tested in ribozymes.
Any combination of the above listed nucleotide modifications, substitutions, or derivatizations, made at the level of the sugar, base, monophosphate groupings or phosphophodiester linkages, may be made in the compounds of the present invention provided that endonuclease activity is not lost.
The compounds of this invention may be incorporated and expressed in cells as a part of a DNA or RNA transfer vector, or a combination thereof, for the maintenance, replication and transcription of the ribozyme sequences of this invention.
Nucleotide sequences encoding the compounds of this invention may be integrated into the genome of a eukaryotic or prokaryotic host cell for subsequent expression (for example as described by Sambrook, 1989). Genomic integration may be facilitated by transfer vectors which integrate into the host genome. Such vectors may include nucleotide sequences, for example of viral or regulatory origin, which facilitate genomic integration. Methods for the insertion of nucleotide sequences into host genome are described for example in Shambrook et al. (1989).
Nucleic acid sequences encoding the ribozymes of this invention and integrated into the genome of a host cell preferably include promoter and enhancer elements operably linked to the nucleotide sequence encoding the ribozyme of this invention, together with an appropriate termination sequence, so as to be capable of expressing said ribozyme in a eukaryotic (such as animal or plant) or prokaryotic (such as bacteria) host cell. Ideally, the promoter and enhancer elements are designed for expression in a tissue and/or developmentally specific manner.
Additionally, the compounds of the present invention may be prepared by methods known per se in the art for the synthesis of RNA molecules, (for example, according to recommended protocols of Promega, Madison, Wis., USA). In particular, the ribozymes of the invention may be prepared from a corresponding DNA sequence (DNA which on transcription yields a ribozyme, and which may be synthesized according to methods known per se in the art for the synthesis of DNA) operably linked to an RNA polymerase promoter such as a promoter for T3 or T7 polymerase or SP6 RNA polymerase. A DNA sequence corresponding to a ribozyme of the present invention may be ligated into a DNA transfer vector, such as plasmid or bacteriophage DNA. Where the transfer vector contains an RNA polymerase promoter operably linked to DNA corresponding to a ribozyme, the ribozyme may be conveniently produced upon incubation with an RNA polymerase. Ribozymes may, therefore, be produced in vitro by incubation of RNA polymerase with an RNA polymerase promoter operably linked to DNA encoding a ribozyme, in the presence of ribonucleotides and an appropriate buffer. In vivo, prokaryotic or eukaryotic cells (including mammalian, plant and yeast cells) may be transfected with an appropriate transfer vector containing genetic material encoding a ribozyme in accordance with the present invention, operably linked to an RNA polymerase promoter such that the ribozyme is transcribed in the host cell. Transfer vectors may be bacterial plasmids or viral (RNA and DNA) or portion thereof. Nucleotide sequences corresponding to ribozymes are generally placed under the control of strong promoters such as the lac, SV40 late, SV40 early, metallothionein, or lambda promoters. Particularly useful are promoters regulated in a tissue or a temporal (developmental) specific manner, or a tightly regulated inducible promoter suitable for gene therapy, which may be under the control of exogenous chemicals. The vector may be an adenovirus or an adeno-associated virus (see for example PCT International Publication No. WO 93/03769, xe2x80x9cAdenovirus Mediated Transfer of Genes to the Gastrointestinal Tractxe2x80x9d, PCT International Publication No. WO 94/11506, xe2x80x9cAdenovirus-Mediated Gene Transfer to Cardiac and Vascular Smooth Muscle,xe2x80x9d PCT International Publication No. WO 94/11522, xe2x80x9cVector for the Expression of Therapy-Relevant Genes,xe2x80x9d PCT International Publication No. WO 94/11524, xe2x80x9cTargetable Vector Particles,xe2x80x9d PCT International Publication No. WO 94/17832, xe2x80x9cTargeting and Delivery of Genes and Antiviral Agents into Cells by the Adenovirus Pentonxe2x80x9d). Ribozymes may be directly transcribed in vivo from a transfer vector, or alternatively, may be transcribed as part of a larger RNA molecule. For example, DNA corresponding to ribozyme sequences may be ligated into the 3xe2x80x2 end of a reporter gene, for example, after a translation stop signal. Larger RNA molecules may help to stabilize the ribozyme molecules against nuclease digestion within cells. On translation, the reporter gene may give rise to a protein, possibly an enzyme whose presence can be directly assayed.
The compounds of this invention may be involved in gene therapy techniques where, for example, cells from a human suffering from a disease, such as HIV, are removed from a patient, treated with the ribozyme or transfer vector encoding the ribozyme to inactivate the infectious agent, and then returned to the patient to repopulate a target site with resistant cells, so called ex vivo therapy. Such cells would be resistant to HIV infection and the progeny thereof would also confer such resistance. In the case of HIV, nucleotide sequences encoding ribozymes of this invention capable of inactivating the HIV virus may be integrated into the genome of lymphocytes or may be expressed by a non-integrating vector such as adenovirus. Alternatively, the nucleotide sequences-may be expressed by a retrovirus or modified retrovirus known for use in the treatment of HIV.
A transfer vector such as a bacterial plasmid or viral RNA or DNA or portion thereof, encoding one or more of the compounds of this invention, may be transfected into cells of an organism in vivo. Once inside the cell, the transfer vector in some cases may replicate and be transcribed by cellular polymerases to produce ribozyme RNAs which may have ribozyme sequences of this invention; the ribozyme RNAs produced may then inactivate a desired target RNA. Alternatively, a transfer vector containing one or more ribozyme sequences may be transfected into cells by electroporation, PEG, high velocity particle bombardment or lipofectants, or introduced into cells by way of micromanipulation techniques such as microinjection, such that the transfer vector or a part thereof becomes integrated into the genome of the host cell. Transcription of the integrated genetic material gives rise to ribozymes, which act to inactivate a desired target RNA. Transfer vectors expressing ribozymes of this invention may be capable of replication in a host cell for stable expression of ribozyme sequences. Alternatively, transfer vectors encoding ribozyme sequences of this invention may be incapable of replication in host cells, and thus may result in transient expression of ribozyme sequences. Methods for the production of DNA and RNA transfer vectors, such as plasmids and viral constructs are well known in the art and are described for example by Sambrook et al. (1989).
Transfer vectors would generally comprise the nucleotide sequence encoding the ribozyme of this invention, operably linked to a promoter and other regulatory sequences required for expression and optionally replication in prokaryotic and/or eukaryotic cells. Suitable promoters and regulatory sequences for transfer vector maintenance and expression in plant, animal, bacterial, and other cell types are well known in the art.
The ribozymes of the present invention have extensive therapeutic and biological applications. For example, disease-causing viruses in man and animals may be inactivated by administering to a subject infected with a virus, a ribozyme in accordance with the present invention adapted to hybridize to and cleave specific RNA transcripts of the virus. Such ribozymes may be delivered by parenteral or other means of administration. Alternatively, a subject infected with a disease causing virus may be administered a non-virulent virus such as vaccinia or adenovirus which has been genetically engineered to contain DNA corresponding to a ribozyme operably linked to an RNA promoter, such that the ribozyme is transcribed in the cells of the host animal, transfected with the engineered virus, to effect cleavage and/or inactivation of the target RNA transcript of the disease causing virus.
The ribozymes of the present invention have particular application to viral diseases caused for example, by the herpes simplex virus (HSV) or the AIDS virus (HIV). Further examples of human and animal disease which may be treated with the ribozymes of this invention include human disorders such as inflammatory diseases (e.g. arthritis) and circulatory disorders (e.g. artheroscierosis and restenosis), psoriasis, cervical preneoplasia, papilloma disease, bacterial and prokaryotic infection, viral infection and neoplastic conditions associated with the production of aberrant RNAs such as occurs in chronic myeloid leukemia. Diseases or infections which may be treated in plants with ribozymes of this invention include fungal infection, bacterial infections (such as Crown-Gall disease) and disease associated with plant viral infection. Of particular interest would be compounds targeting genes associated with male gametophyte development, (examples include PCT International Publication No. WO 92/18625, entitled xe2x80x9cMale-Sterile Plants, Method For Obtaining Male-Sterile Plants And Recombinant DNA For Use Thereinxe2x80x9d; U.S. Pat. No. 5,254,802, entitled xe2x80x9cMale Sterile Brassica Plantsxe2x80x9d;PCT international Publication No. WO 93/25695, entitled xe2x80x9cMaintenance of Male-Sterile Plantsxe2x80x9d;PCT International Publication No. WO 94/25593, entitled xe2x80x9cMethod For Obtaining Male-Sterile Plants; PCT International Publication No. WO 94/29465, entitled xe2x80x9cProcess For Generating Male Sterile Plantsxe2x80x9d).
The period of treatment would depend on the particular disease being treated and could be readily determined by a physician or by a plant biologist as appropriate. Generally treatment would continue until the disease being treated was ameliorated.
The ribozymes of the present invention also have particular application to the inactivation of RNA transcripts in bacteria and other prokaryotic cells, plants, animals and yeast cells. In bacteria, RNA transcripts of, for example, bacteriophage (which cause bacterial cell death) may be inactivated by transfecting a cell with a DNA transfer vector which is capable of producing a ribozyme in accordance with the present invention which inactivates the phage RNA. Alternatively, the ribozyme itself may be added to and taken up by the bacterial cell to effect cleavage of the phage RNA. Similarly, eukaryotic and prokaryotic cells in culture may, for example, be protected from infection or disease associated with mycoplasma infection, phage infection, fungal infection and the like.
RNA transcripts in plants may be inactivated using ribozymes encoded by a transfer vector such as the Ti plasmid of Agrobacterium tumefaciens. When such vectors are transfected into a plant cell and integrated, the ribozymes are produced under the action of RNA polymerase and may effect cleavage of a specific target RNA sequence. Endogenous gene transcripts in plant, animal or other cell types may be inactivated using the compounds of the present invention. Accordingly, undesirable phenotypes or characteristics may be modulated. It may, for example, be possible using the ribozymes of the present invention to remove stones from fruit or treat diseases in humans which are caused by the production of a deleterious protein, or over-production of a particular protein. The compounds described above may be used to effect male sterility by destroying the pollen production in a plant. Furthermore, for the in vivo applications of the ribozymes of this invention in humans, animals, plants, and eukaryotic and prokaryotic cells, such as in phenotypic modification and the treatment of disease, it is necessary to introduce the ribozyme into cells whereafter, cleavage of target RNAs takes place. In vivo applications are highly suitable to the compounds as discussed herein. Methods for the introduction of RNA and DNA sequences into cells, and the expression of the same in prokaryotic and eukaryotic cells are well known in the art. The same widely known methods may be utilized in the present invention.
The compounds of this invention may be incorporated into cells by direct cellular uptake, where the ribozymes of this invention would cross the cell membrane or cell wall from the extracellular environment. Agents may be employed to enhance cellular uptake, such as liposomes or lipophilic vehicles, cell permeability agents, such as dimethylsulfoxide, and the like.
The compounds of the present invention may be combined with pharmaceutically and veterinarally acceptable carriers and excipients which are well known in the art, and include carriers such as water, saline, dextrose and various sugar solutions, fatty acids, liposomes, oils, skin penetrating agents, gel forming agents and the like, as described for example in Remington""s Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.
Agriculturally acceptable carriers and excipients are well known in the art and include water; surfactants; detergents; particularly biodegradable detergents; talc; inorganic and/or organic nutrient solutions; mineral earths and clays; calcium carbonate; gypsum; calcium sulfate; fertilizers such as ammonium sulfate, ammonium phosphate, urea, carborundum, and Agrobacterium tumefaciens; and natural products of vegetable origin such as, for example, grain, meals and flours, bark meals; and the like.
The compounds of this invention may be provided in a composition with one or more anti-viral, anti-fungal, anti-bacterial, anti-parasitic, anti-protozoan or anthelminthic agents, herbicides, pesticides or the like, for example as described in the Merck Index (1989) 11th Edition, Merck and Co. Inc.
By way of example only, therapeutic compositions of this invention may be directed against Herpes Simplex virus types 1 and 2, psoriasis, cervical preneoplasia, papilloma disease, and bacterial and prokaryotic infection. Such treatments may, for example, involve topical application of ribozyme to the site of disease. For example, in the treatment of Herpes virus lesions, ribozymes may be formulated into a cream containing a concentration of 0.1 nM to 100 mM ribozyme, preferably 1 nM to 1 mM. The cream may then be applied to the site of infection over a 1 to 14 day period in order to cause amelioration of symptoms of the infection. Prior to the final development of topical formulations for the treatment of virus infection, effectiveness and toxicity of the ribozymes and formulations involving them may, for example, be tested on an animal model, such as scarified mouse ear, to which virus particles, such as 2xc3x97106 plaque forming units are added. A titer of infectious virus particles in the ear after treatment can then be determined to investigate effectiveness of treatment, amount of ribozyme required and like considerations. Similar investigations in animal models prior to human trials may also be conducted, for example, in respect of the treatment of psoriasis, papilloma disease, cervical preneoplasia, and in diseases such as HIV infection, bacterial or prokaryotic infection, viral infection and various neoplastic conditions which involve a deleterious RNA species.
Compositions for topical application are generally in the form of creams, where the ribozymes of this invention may be mixed with viscous components. The compounds of this invention may be incorporated into liposomes or other barrier type preparations to shield the ribozymes from nuclease attack or other degradative agents (such as nucleases and adverse environmental conditions such as UV light).
Compositions may be provided as unit dosages, such as capsules (for example gelatin capsules), tablets, suppositories and the like. Injectable compositions may be in the form of sterile solutions of ribozyme in saline, dextrose or other media. Compositions for oral administration may be in the form of suspensions, solutions, syrups, capsules, tablets and the like. Ribozymes may also be provided in the form of an article for sustained release, impregnated bandages, patches and the like. The compounds of this invention may be embedded in liposomes or biodegradable polymers such as polylactic acid. Pharmaceutical compositions which may be used in this invention are described, for example, in Remington""s Pharmaceutical Sciences, see above.
The present invention is further directed to a plant DNA expression cassette comprising a gene sequence flanked by promoter and terminator sequences at its 5xe2x80x2 and 3xe2x80x2 ends respectively wherein said genetic sequence on expression provides a ribozyme RNA. The DNA cassette may further be part of a DNA transfer vector suitable for transferring the DNA cassette into a plant cell and insertion into a plant genome. In a most preferred embodiment of the present invention, the DNA cassette is carried by broad host range plasmid which is capable of transformation into plant cells using Agrobacterium comprising Ti DNA on the left and right borders, a selectable marker for prokaryotes, a selectable marker for eukaryotes, a bacterial origin of replication and optional plant promoters and terminators. The present invention also includes other means of transfer such as genetic bullets (e.g. DNA-coated tungsten particles, high-velocity micro projectile bombardment) and electroporation amongst others.
The present invention is also directed to a transgenic plant resistant to a virus, its genome containing a sequence which gives rise, on transcription, to the nucleic acid molecule mentioned above. This transgenic plant, including fruits, and seeds thereof, may be from alfalfa, apple, arabidopsis, barley, bean, canola (oilseed rape), cantaloupe, carnation, cassava, casuarina, clover, corn, cotton, courgette, cucumber, eucalyptus, grape, melon, papaya, pepper, potato, rice, rose, snap dragon, soybean, squash, strawberry, sunflower, sweet pepper, tobacco, tomato, walnut, wheat or zucchini. Also included are the plant cells transformed by the above-mentioned transfer vector comprising a nucleotide sequence which is, or on transcription gives rise to, the nucleic acid molecule.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, and or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.